Neurodegeneration can be triggered by a variety of genetic, epigenetic, and environmental factors. Healthy neurons are able to maintain their integrity throughout the life of an animal, suggesting the existence of a maintenance mechanism that allows neurons to sustain, mitigate or even repair damage. Previous work in our lab and others has found NMNAT proteins in Drosophila and mammals to be robust and versatile neuroprotective factors. However, it remains unclear whether and how neurons regulate the neuroprotective processes mediated by maintenance factors such as NMNAT. How neurons partition NMNAT into two distinct functions ? NAD metabolism and neuroprotection, and how such partitioning is regulated under normal and adverse conditions to achieve neuroprotection are the focus of this proposal. Our preliminary studies have found that Drosophila nmnat is alternatively spliced, producing splice isoforms with divergent neuroprotective activities, and that neurons could alter the preference for splice variants production under stress towards the neurprotective variant and therefore achieve self-protection. We hypothesize that alternative splicing and other post-transcriptional regulations are an essential endogenous program for regulating neuronal self-protective response under normal, stress, and disease conditions. Importantly this mechanism is likely conserved between fly and mammals as we have collected preliminary data indicating that human NMNAT genes are also alternatively spliced. In this proposal, we plan to uncover the key mechanisms to enhance neuroprotection in Drosophila and then extend the study to human NMNATs in mammalian neurons. First, we will characterize the biochemical and cellular properties of Drosophila and human NMNAT protein isoforms, and illustrate the role of alternative splicing in post-transcriptional regulation of nmnat genes. Next, we will identify alternative splicing as a stress response that affords protection to neurons, and further reveal the functional role of microRNAs in regulating the abundance of neuroprotective RNA variants and modulating the neuroprotective efficacy of NMNAT. Finally, we will study human NMNAT protein isoforms and characterize the neuroprotective function of alternatively spliced NMNAT isoforms in cultured DRG explants and in vivo in corticospinal track (CST) neurons, and identify key mechanisms underlying the divergent neuroprotective effects of NMNAT variants in degenerative conditions. Studies on Drosophila and human NMNATs using a ?two-model? approach will reveal the evolutionarily conserved posttranscriptional regulatory mechanisms and identify strategies relevant to enhancing neuroprotection in humans.